Abstract
ObjectivesTo investigate the role of EphA2 in malignant cellular behavior in renal cell carcinoma (RCC) cells and whether FAK/RhoA signaling can act as downstream effectors of EphA2 on RCC cells.MethodsExpression of EphA2 protein in non-metastatic RCC (Caki-2 and A498), metastatic RCC cells (Caki-1 and ACHN), HEK-293 cells and prostate cancer cells (PC-3 and DU-145; positive controls of EphA2 expression) was evaluated by Western blot. Changes in mRNA or protein expression of EphA2, FAK or membrane-bound RhoA following EphA2, FAK or RhoA small interfering RNA (siRNA) transfection were determined by reverse transcription polymerase chain reaction or Western blot. The effect of siRNA treatment on cellular viability, apoptosis and invasion was analyzed by cell counting kit-8, Annexin-V and modified Matrigel-Boyden assays, respectively.ResultsIn all RCC cell lines, the expression of EphA2 protein was detectable at variable levels; however, in HEK-293 cells, EphA2 expression was very low. Treatment with EphA2 siRNA significantly reduced the expression of EphA2 mRNA and protein in all RCC cell lines. For non-metastatic RCC cells (Caki-2 and A498) but not metastatic RCC cells (Caki-1 and ACHN), cellular viability, invasiveness, resistance to apoptosis, expression of membrane-bound RhoA protein and FAK phosphorylation were significantly decreased in EphA2 siRNA-treated cells compared to the control. In non-metastatic RCC cells, FAK siRNA significantly attenuated the invasiveness, resistance to apoptosis, as well as expression of membrane-bound RhoA protein without changing protein expression of EphA2. RhoA siRNA significantly decreased the malignant cellular behavior and expression of membrane-bound RhoA protein without changing EphA2 protein expression or FAK phosphorylation.ConclusionsOur data provide the first functional evidence that the EphA2/FAK/RhoA signaling pathway plays a critical role in the malignant cellular behavior of RCC and appears to be functional particularly in the early stage of malignant progression of non-metastatic RCC.
Highlights
25% of patients with renal cell carcinoma (RCC) present distant metastases at diagnosis and approximately 30% of RCC patients eventually develop metastases during the disease course [1]
In all RCC cell lines, the expression of EphA2 protein was detectable at variable levels; in human embryonic kidney-293 (HEK-293) cells, EphA2 expression was very low
For non-metastatic RCC cells (Caki-2 and A498) but not metastatic RCC cells (Caki-1 and ACHN), cellular viability, invasiveness, resistance to apoptosis, expression of membranebound RhoA protein and Focal adhesion kinase (FAK) phosphorylation were significantly decreased in EphA2 small interfering RNA (siRNA)-treated cells compared to the control
Summary
25% of patients with renal cell carcinoma (RCC) present distant metastases at diagnosis and approximately 30% of RCC patients eventually develop metastases during the disease course [1]. Advanced RCC is highly refractory to conventional therapy including radiation and chemotherapy, and the effectiveness of immunotherapy is still controversial [2]. Targeted therapy such as tyrosine kinase inhibitors and mammalian target of rapamycin (mTOR) inhibitors have been introduced recently, but there are no data to indicate that it is curative, and most patients who undergo this therapy eventually relapse, leading to death from RCC [3]. EphA2, a member of the Eph family, is overexpressed in tumor cells of various types of cancer including breast, prostate and colon [5]. A previous study showed that higher levels of EphA2 expression were correlated with higher grades of RCC and could be a risk factor for accelerated disease recurrence and indicative of a poor prognosis in surgically treated patients with RCC [6]
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